Use of the salivary protein CD14 as an indicator of the low risk to developing dental caries

ABSTRACT

It is generally accepted that salivary components are important for dental health, but till now no clear correlation has been found between one or more of said components and the onset of dental caries. The present invention relates to an assay method comprising analysing the presence and/or the content of the salivary soluble CD14 protein from a saliva sample of the individual subjected to examination; the absence of said protein from the salivary sample, or its presence in a reduced amount compared to a predetermined threshold value in caries-free individuals, is considered as a marker of susceptibility to developing caries and/or as a diagnostic element for the existence of ongoing carious lesions.

FIELD OF THE INVENTION

The present invention relates to an in vitro assay method useful fordetermining the receptivity of an individual to caries, particularly toa method for determining the predisposition of an individual todeveloping dental caries or for determining the presence of one or moreactive caries within the oral cavity.

BACKGROUND OF THE INVENTION

Caries is a multifactorial infectious disease, determined by the dynamicbalance between pathological factors leading to demineralization andprotective factors leading to remineralization (Featherstone, 2004).

Many salivary proteins have been suggested to play a role as eithercariogenic or anti-cariogenic factors, depending on their ability toinhibit the growth of acid-producing bacteria, to aggregate them, topromote their adhesion to the tooth surface or to modify themineralization/demineralization balance (Lenander-Lumikari andLoimaranta, 2000; Nieuw Amerongen et al., 2004).

Understanding the role of salivary proteins in caries development ismade more complicated by the fact that many of these proteins aremultifunctional (the same protein may perform different functions),redundant (many proteins seem to share the same functions) andamphifunctional (the same protein may have opposite effects, dependingon the intraoral environment) (Rudney, 2000; Humphrey and Williamson,2001).

No significant differences in the protein composition of parotid saliva(Dodds et al., 1997) and whole saliva (Banderas-Tarabay et al., 2002)were detected between caries-active (CA) and caries-free (CF)individuals.

Actually, each band of one-dimensional gel electrophoresis is oftencomposed of different protein types, thus making it difficult to detectquantitative changes in specific proteins. The mapping of human salivaryproteins by two-dimensional gel electrophoresis has revealed thepresence of many spots which are still to be identified (Ghafouri etal., 2003): one of these proteins could turn out to be primarilyinvolved in the development of caries.

However, the prior art does not contain any indication as to thepossible existence of any specific salivary protein which may be used asa marker for the predisposition of an individual to developing caries oras an indicator of the presence of ongoing caries.

SUMMARY OF THE INVENTION

The present invention is based on the recognition of the fact that theexpression of the salivary protein sCD14 (soluble CD14) is considerablyreduced in the saliva from CA individuals compared to the saliva of CFindividuals; in particular, by Western blotting analysis of salivarysamples from young CA patients, the sCD14 protein was determined to beabsent from all of the salivary samples from young CA patients, whilstit was clearly detectable in the saliva from all of the controlage-matched CF individuals.

DETAILED DESCRIPTION OF THE INVENTION

The inverse relationship between the presence of sCD14 in the saliva andthe onset of early tooth decay leads to suppose that this salivaryprotein may play an important role in preventing the development ofcaries or that its disappearance (or clear-cut reduction) may representa marker for the presence of active caries.

Thus, the subject-matter of the present invention comprises a prognosticand diagnostic assay method for determining the predisposition of anindividual to developing dental caries or for detecting the existence ofactive caries within the oral cavity, characterized in that it comprisesthe step of examining a salivary sample from the individual for thepresence of the salivary soluble CD14 protein, wherein the absence ofsaid protein from the sample or its presence in a reduced amountcompared to a predetermined threshold value in caries-free individuals,is indicative of said predisposition or of the presence of activecaries.

CD14 is a 55 kDa membrane glycoprotein expressed predominantly on thesurface of monocytes/macrophages and neutrophils, which plays a crucialrole in the recognition of several microbial products, such aslipopolysaccharide (LPS, endotoxins) and peptidoglycan, which are majorcomponents of Gram-negative and Gram-positive bacteria, respectively,thus participating in the initiation of immune responses (Lien andIngalls, 2002).

The LPS- or peptidoglycan-CD14 complex, together with other accessoryproteins, interacts with cell surface receptors designated as Toll-likereceptors, and through the activation of multiple signalling pathwaysleads to the synthesis of pro-inflammatory cytokines (Guha and Mackman,2001).

CD14 is expressed on the cell surface via a glycosylphosphatidylinositolanchor, but is also found in a free form in plasma, this being referredto as soluble CD14 (sCD14). sCD14 mediates the activation by LPS ofCD14-negative cells, such as endothelial and epithelial cells (Frey etal., 1992).

Major human salivary glands constitutively express and secrete sCD14into saliva (Sugawara et al., 2002): salivary sCD14 may mediate theactivation of CD14-lacking intestinal epithelial cells by LPS via theToll-like receptor TLR4 (Uehara et al., 2003), and promotes the invasionof oral epithelial cells by Actinobacillus actinomycetemcomitans,thereby increasing the production of interleukin-8 (Takayama et al.,2003). Although this may suggest that human sCD14 could play animportant role in the innate immunity in the oral cavity, there is noevidence in the prior art as to a correlation between salivary sCD14 andthe receptivity of an individual to caries.

The results obtained within the present invention show that the saidprotein, besides its multifunctional properties, plays an important roleas an anti-cariogenic factor.

A number of hypothesis may be formulated to explain such a function,although the present invention is not intended to be bound norrestricted to any particular explanation of the mechanism of action.

First, sCD14 could enable oral epithelial cells to bind bacteria(thereby preventing their adhesion to the tooth surface) and to producecytokines capable of recruiting phagocytes at the gingiva-toothinterface (thereby enhancing the clearance of microbes from saliva).

Undoubtedly, CD14-lacking epithelial cells respond to LPS in ansCD14-dependent manner to produce interleukin-8, which inducesactivation and migration of neutrophils (Uehara et al., 2001).

sCD14 could also modulate the anti-inflammatory properties of saliva,binding with high affinity to human lactoferrin (Baveye et al., 2000),an iron- and LPS-chelating glycoprotein found in exocrine secretions ofmammals (Caccavo et al., 2002).

Furthermore, the binding of sCD14 to bacteria could hinder theirtransition from the planktonic to the sessile phase, retarding plaqueformation.

The analysis of the salivary sample for the determination of sCD14within said sample may be carried out by Western blotting analysis whichis known per se; to this end, primary anti-human CD14 antibodies arecommercially available, for example the goat polyclonal anti-human CD14antibody, from Upstate, D.B.A., Italy.

ELISA kits useful for qualitative and quantitative determination ofhuman soluble CD14 are also commercially available.

The following examples illustrate the analyses carried out within thescope of the present invention.

Example 1 Salivary Analysis

40 unrelated Italian healthy children, aged 6 to 12 years, were examinedclinically by an experienced dentist; criteria for inclusion were nogeneral diseases, no medication and no extracted teeth. 20 of them (8males, 12 females; age=8.45+0.358 years) were caries-free (CF), and 20(9 males, 11 females; age=7.9+0.341 years) were caries-active (CA:defined as having from 2 to 8 carious lesions requiring restoration).

The study was approved by the local ethical committee; written and oralinformed consent was given by all participants.

The subjects were instructed to refrain from eating, drinking or usingbreath fresheners for a minimum of 2 hours before collecting saliva.Unstimulated human whole saliva (about 5 ml) was collected between 8 and10 a.m. by the same examiner prior to clinical examination, to reducepossible circadian variations.

The subjects were invited to clean their teeth with a toothbrush,rinsing the mouth with water. After a 10 min waiting time, subjects wereasked to spit out saliva into sterile plastic tubes, which wereprocessed immediately thereafter, to minimize proteins degradation(Banderas-Tarabay et al., 2002).

The samples were supplemented with the protease inhibitor cocktail setIII (100 mM AEBSF, 80 μM aprotinin, 5 mM bestatin, 1.5 mM E-64, 2 mMleupeptin, and 1 mM pepstatin; Calbiochem-Novabiochem Corporation, CA)and kept on ice throughout the processing. Saliva was centrifuged at12,000 rpm for 15 min at 4° C. to remove insoluble material, cells anddebris (Ghafouri et al., 2003). 2 ml of the sample were desalted by gelfiltration (PD-10 column, Amersham International, Piscataway, N.J.) in12 mM ammonium bicarbonate, pH 7.1. Eluates were lyophilized and storedat 70° C. until electrophoretic analysis.

Example 2 Western Blot Analysis

Electrophoresis reagents were from Bio-Rad Laboratories (Richmond,Calif.). The protein content of whole saliva was assessed with the BCAkit from Pierce (Rockford, Ill.).

If not otherwise specified, other reagents were purchased from SigmaChemical Co. (St. Louis, Mo.) and Aldrich (Milan, Italy).

Samples were directly solubilized in lysis buffer (125 mM Tris-HCl, 4%sodium dodecylsulfate, 20% glycerol, pH 6.8, 10% β-mercaptoethanol, and0.002% bromophenol blue) and boiled for 5 min prior to running gels.Aliquots containing 30 μg of proteins were subjected to sodiumdodecylsulfate-polyacrylamide gel electrophoresis (12% polyacrylamide).Proteins were transferred to a PVDF filter membrane (Immobilon P,Millipore, Bedford, Mass.) and incubated with an anti-human CD14polyclonal antibody (from goat; Upstate, D.B.A., Italy), diluted 1:500in PBS-BSA 1%.

After overnight incubation, the membrane was washed with PBS-Tween 0.1%and subjected for 1 h to a peroxidase-conjugated anti-goat IgG antibody(from mouse; Amersham International), diluted 1:1000 in PBS-Tween withBlocker Non-Fat Dry Milk 5% (Bio-Rad, Calif.). The PVDF membrane waswashed again with PBS-Tween and proteins were detected by enhancedchemiluminescence (Amersham International).

Molecular-weight standards were used in all gels and the density of theprotein bands was quantitated with a Gel-Doc densitometer (Bio-Rad).

FIG. 1 illustrates the expression of soluble CD14 in human whole saliva,as determined from the above-illustrated experimental tests: A is theWestern blot analysis on saliva samples obtained from the 20 CF subjects(samples 1 to 20) and from the 20 CA subjects (samples 21 to 40); Billustrates the densitometric quantification of the bands obtained fromsubjects 1-20.

It can be observed that the immunoblot analysis shows the presence ofthe 55 kDa soluble form of CD14 in all of the CF subjects and itsabsence from all of the CA subjects.

According to the invention, the absence of sCD14 in a Western blotanalysis may be considered as a marker of the predisposition of theindividual subjected to examination to developing dental caries or as adiagnostic marker of ongoing caries; furthermore, the determination of areduced sCD14 expression in the salivary samples compared to apredetermined threshold value in caries-free subjects, is to beconsidered as predictive of the development of dental caries or as adiagnostic marker of ongoing caries.

For this purpose, the quantitative analysis of sCD14 is preferablycarried out by ELISA assay. The determination of the threshold value ispreferably carried out by statistical analysis of healthy CF subjects,preferably age-matched with the individual subjected to analysis. Inparticular, the predictivity of the test is to be considered asparticularly high when the value of expression of salivary sCD14 islower than the 20% of the predetermined threshold value.

The onset of caries in infancy and youth is strongly suggestive of theinvolvement of genetic factors predisposing to a faster tooth decay.Since several polymorphisms in the 5′ flanking region of the CD14 genehave been associated with higher levels of serum sCD14 (Baldini et al.,1999; Vercelli et al., 2001), the reduced salivary expression of sCD14in young patients with multiple caries is expected to be relatable to aspecific polymorphism of the gene.

In that respect, the sCD14 polymorphisms can be studied with theRFLP-PCR technique, which allows to detect every single mutatednucleotide, based on the analysis of the different electrophoreticpattern obtained from the fragment resulting from the cleavage byspecific restriction enzymes of the previously PCR-amplified DNA.

Therefore, investigation as to a specific polymorphism of theCD14-encoding gene as a marker of genetic susceptivity to thedevelopment of cariesfalls within the scope of the present invention.

REFERENCES

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1. A method for diagnosing existence of dental caries in an individual,comprising the steps of: obtaining a salivary sample from theindividual; and analysing by Western Blotting or ELISA said salivarysample from the individual to determine the presence or absence ofsalivary soluble CD14 protein in said salivary sample, wherein absenceof said salivary soluble CD14 protein in said salivary sample isindicative of the existence of dental carries in the individual.
 2. Amethod for diagnosing existence of dental caries in an individual,comprising the steps of: obtaining a salivary sample from theindividual; analysing by Western Blotting or ELISA said salivary samplefrom the individual for the presence of salivary soluble CD14 protein insaid salivary sample; obtaining a quantitative value of expression ofsaid salivary soluble CD14 protein in said salivary sample; comparingsaid quantitative value of expression of said salivary soluble CD14protein in said salivary sample from the individual with a predeterminedvalue of the expression of said salivary soluble CD14 protein obtainedfrom statistical analysis by ELISA of a salivary sample from individualsknow to be free of dental carries, wherein a reduced quantitative valueof expression of said salivary soluble CD14 protein in said salivarysample from the individual compared with said predetermined value isindicative of the existence of dental carries in the individual.
 3. Theassay method according to claim 2, wherein the existence of dentalcaries is determined as positive when said quantitative value ofexpression of salivary soluble CD14 in said salivary sample subjected toanalysis is at least 20% lower than said predetermined quantitativevalue.
 4. A method for diagnosing existence of dental caries in anindividual, comprising the steps of: obtaining a salivary sample fromthe individual; and analysing by Western Blotting or ELISA said salivarysample from the individual to detect the salivary soluble CD14 proteinin said salivary sample, wherein non-detection of said salivary solubleCD14 protein in said salivary sample is indicative of the existence ofdental carries in the individual.